PROJECT SUMMARY Studies to delineate the autoimmune-promoting effects of the environmental pollutant, trichloroethylene (TCE), have focused on functional effects in effector/memory CD4+ T cells from lupus-prone mice. Our long-term goal is to understand how TCE drives CD4 cell differentiation to promote autoimmunity and/or hypersensitivity disorders. The objectives are (i) to determine how TCE through its primary metabolite, trichloroacetaldehyde hydrate (TCAH), alters differentiation of nave CD4 cells to effector/memory subsets important in promoting (e.g., Th1 and Th17) or suppressing autoimmunity (e.g., Th2 and TREG) in vitro and (ii) to determine pathogenicity of these subsets in vivo. CD4s will be compared in both autoimmune-prone (MRL+/+) and resistant (B6) strains of mice to understand the contribution of genetic susceptibility factors. Inclusion of both sexes may reveal why being female elevates autoimmune disease risk. The central hypothesis is that TCAH promotes the differentiation of pathogenic effector cells and/or decreases expansion of effector cells associated with suppression of autoimmunity involving changes in gene expression and DNA methylation. TCAH will generate unique genetic signatures although immune pathology will be observed in MRL+/+ mice rather than in B6 mice, that will be more robust in females vs. males. The rationale is that it is now apparent that key CD4 functional effects that may be regulated at the level of DNA methylation occur during differentiation, and thus cannot be studied in already differentiated cells. The central hypothesis will be tested in three specific aims: 1) Define effects of TCAH on CD4+ T cell differentiation by assessing gene expression and 2) DNA methylation and 3) Determine pathogenicity of differentiating Th subsets in vivo. Under the first Aim, RNA-Seq will determine whether TCAH directly alters the expression of genes that may confer a selective advantage to pathogenic effector CD4+ T cells. In Aim 2, reduced representation bisulfite sequencing will be used to determine TCAH-induced changes in DNA methylation in differentiating CD4+ T cells. Experiments will test whether gene expression changes induced by TCAH are mediated at the level of DNA methylation using either methyl donors or methylation inhibitors to reverse effects. The presence of important methyl variants will be determined by flow cytometry. The third Aim will test Th subsets for their ability to generate immune pathology in adoptive transfer experiments. Treatment of cells with methyl donors prior to transfer will connect Aim 1 and 2 results to determine whether TCAH promotes T cell-mediated pathology in a DNA methylation- dependent manner. The proposed research is innovative and significant because it focuses on how TCAH alters the fluid DNA methylation pattern generated during differentiation that will lead to the discovery of novel gene or methylation patterns that may be responsible for TCE-induced immune disorders in humans. Such knowledge may identify immune-mediated pathways for targeted therapy by normalizing immune responses in TCE-exposed individuals.